Prostaglandin E2 (PGE2) is a bioactive lipid signaling molecule implicated in a range of inflammatory diseases and cancers. In most human cells, PGE2 is biosynthesized via sequential oxidation of arachidonic acid (AA) by cyclooxygenases (COX) and prostaglandin E2 synthase (PTGES). At least two fungi that are pathogenic to humans also produce PGE2, which is thought to potentiate their invasive properties in humans. Our preliminary studies show that Saccharomyces cerevisiae (brewers yeast), a saprophytic fungus, also produces PGE2. PGE2 generated by S. cerevisiae is of concern because this fungus is used to manufacture a wide range of products including alcoholic beverages and recombinant proteins for therapeutics and vaccines. We investigated PGE2 levels in alcoholic beverages using monoclonal antibodies having high specificity for PGE2 and detected PGE2 in beer and sake. Moreover, in both products, we detected live yeast that biosynthesize PGE2 when provided AA as a substrate. However, the sequenced genomes of fungi, including S. cerevisiae, lack genes homologous to mammalian COX and PTGES, suggesting a novel biochemical pathway for PGE2 biosynthesis by fungi. Because PGE2 and/or PGE2 synthesizing yeast that are consumed by humans may stimulate or promote gastrointestinal (GI) disease, we propose to characterize PGE2 levels in products manufactured using S. cerevisiae with liquid chromatography-mass spectrometry. To determine the potential consequences of PGE2 on cells of the GI tract, we aim to examine the biological responses of cultured cells derived from the GI tract to the quantities of PGE2 found in products manufactured using S. cerevisiae with quantitative reverse transcription PCR. The fungal PGE2-synthesis pathway merits investigation because of its biochemical uniqueness and the potential of fungal PGE2 modulating disease in humans. Thus, we propose to define the fatty acid (FA) substrates and enzymes responsible for PGE2 biosynthesis in S. cerevisiae using genetic, biochemical and MALDI-TOF/TOF mass spectrometry methodologies. We anticipate this research has the potential to improve human health by providing a strategy to limit the introduction of FA substrates for PGE2 into manufacturing processes and point to a means for genetically engineering fungi of economic import which lack key PGE2-synthesis enzymes.